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the different method for bacterial staining
Typology: Lecture notes
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Bacterial Staining Techniques I I. Complete Lab 1 II. Staining Microorganisms: Simple Stains (Direct and Negative) III. Morphological Unknown I. Complete Lab 1: Collect your plates from the trays on the side bench. Observe the TSA plates for colonies of various sizes, shapes and colors. Each bacterial or fungal species gives a characteristic colony color and morphology. Draw the colonies observed on both TSA plates in the spaces provided in the Results section of Lab #1. Pick three colonies from either of the TSA plates and describe the colony color and morphology. Also observe the cloudiness (turbidity) of your nutrient broth tube and estimate the number of bacteria per mL (see turbidity table below). TERMS AND DEFINITIONS Colony : a single cell divides exponentially forming a small, visible collection of cells. Colonies are observed when bacteria are grown on a solid medium. Each colony usually contains 10^7 - 108 bacteria. Colony morphology : Characteristics of a colony such as shape, edge, elevation, color and texture. Turbidity : cloudy appearance of a liquid medium due to the presence of bacteria. You can "estimate" the number of bacteria per mL by using the table below. Turbidity # Bacteria per mL none 0 - 106 light 107 moderate 108 *heavy 109 *Usually bacterial populations do not exceed 3 x 10^9 bacteria/mL when grown in liquid media. II. STAINING MICROORGANISMS A. Smear preparation Simple Stains: B. Direct stain C. Negative stain TERMS AND DEFINITIONS Chromophores : Groups with conjugated double bonds that give the dye its color. Direct , cationic , basic or positive dyes: contain positively charged groups. Examples include methylene blue, basic fuchsin, and crystal violet. These dyes directly bind to and stain the negatively charged surface of bacterial cells. Negative, anionic, or acidic dyes: contain functional groups that have a negative charge. Examples include eosin, nigrosin and Congo red. These dyes are repelled by the negatively charged surface of bacterial cells. Thus, they stain the background, leaving the bacterial cells clear and bright against a dark background. Heat Fixation: application of heat to a bacterial smear preparation. This procedure simultaneously kills and attaches the bacteria to the slide.
A. Smear Preparation The first step in most bacterial staining procedures is the preparation of a smear. In a smear preparation, cells from a culture are spread in a thin film over a small area of a microscope slide, dried, and then fixed to the slide by heating or other chemical fixatives. A good smear preparation should be...
f. After examining the slide - move the oil immersion objective away from the slide. Clean the objective thoroughly with lens paper (NOT KimWipes!) and lens cleaning solution.
III. Morphological Unknown The staining procedures introduced in Labs 2-4 are commonly used by microbiologists to help characterize and identify bacteria. These stains often make it possible to determine the group of organisms to which an unknown isolate belongs. With few exceptions, staining is the first step in identifying a bacterial unknown. Although staining alone does not give sufficient information about the organism to make a definitive identification, it will give some important clues. You will be given an unknown pure culture on which you will perform the various stains as you go through labs 2-4. PROCEDURE: (EACH STUDENT)
Direct Stain: Draw and label examples of Escherichia coli and S. cerevisiae. Be sure to illustrate the relative sizes of each microorganism. Negative Stain QUESTIONS:
**HELPFUL HINT**
You may find it useful to make index cards for each organism, staining procedure, and media encountered throughout this course. At this point, you can record the Gram reaction, morphology, and arrangement for each organism. Later in the course, it will be possible to add further descriptions. The course web site has several features that will assist you in this task. Although it is common practice to abbreviate the genus by using the first letter (e.g. E. coli for Escherichia coli ), for the purpose of this class you will need to write out the entire genus and species name on practical exams and quizzes. The first letter of the genus is always capitalized, but the species is not. Also, the genus and species name is always typed in italic or underlined. Organisms introduced in this lab: Escherichia coli : G- bacilli (rod) Saccharomyces cerevisiae : (yeast) Bacillus subtilis : G+ bacilli